Control of protein synthesis (translation) is vital for cell proliferation and differentiation. In human, initiation of translation is a multi-step process that involves two key GTP hydrolysis steps. Translation initiation relies on a dynamic network of interactions among ribosomes, RNAs and proteins within a mega-dalton translation initiation complex. This is particularly true for the two GTPase steps: start codon selection and ribosomal subunit joining, where a number of interactions are cooperative while others are competing with each other. We hypothesize that synergistic interactions among components of the initiation complex ensure their recruitment to the complex, their proper positioning, as well as the stability of the initiation complex as a whole. We propose that pairs of competing interactions are responsible for remodeling of the initiation complex, whereby one set of interactions are replaced by another set of interactions as the complex matures toward the formation of an active ribosome ready to start synthesizing protein. To test these hypotheses, we propose a multidisciplinary innovative approach, which will use a combination of NMR, fluorescence spectroscopy and other biophysical and biochemical methods. 1. We will use biophysical assays and NMR to determine which interactions among the proteins responsible for start codon selection and ribosomal subunit joining are cooperative and which are competitive and to characterize structurally the binding interfaces involved. 2. We will use steady-state fluorescence anisotropy, time-resolved fluorescence anisotropy decay and biochemical assays on in vitro reconstituted translation initiation complexes to elucidate the temporal regulation of the recruitment of proteins to the initiation complex and their release. 3. We will use Fluorescence Resonance Energy Transfer (FRET) to determine the positions and mutual orientations within the initiation complex of the proteins responsible for start codon selection and ribosomal subunit joining. Proteins with known positions will serve as reference. Together, the results from these aims will provide a comprehensive understanding of the coordination and regulation of start codon selection and ribosomal subunit joining. The long term goals of this proposal are to elucidate the structural organization of the human translation initiation complexes, as well as the mechanisms of their assembly and remodeling. This work will identify key steps in the translation initiation process that are promising targets in manipulating the rates of protein synthesis for therapeutic purposes. PUBLIC HEALTH RELEVANCE: Actively dividing cells, such as cancer cells, require increased rates of protein synthesis and inhibitors of protein synthesis have proved effective in cancer therapy. The work outlined in this proposal will establish new targets for direct and specific inhibition of protein synthesis for use in cancer therapy.